The ability to regulate gene expression is desirable in a variety of situations, including in the production of recombinant proteins, in gene therapy, and in analyses of cell development and differentiation. A wide variety of gene regulation systems have been described, some of which stimulate gene expression in a constitutive manner and some of which stimulate gene expression in an inducible manner. A popular approach to regulating gene expression is to create a transcriptional activator fusion protein (also referred to herein as a "transactivator") which is composed of a DNA binding domain, which has specificity for a particular target DNA binding site, and a transcriptional activation domain. To regulate expression of a gene of interest, the gene is operatively linked to the target DNA binding site and then both the gene and an expression vector encoding the transactivator fusion protein are coexpressed in a host cell. Upon binding of the transactivator fusion protein to the target DNA binding site, expression of the gene of interest is stimulated.
A constitutive transcriptional activator is created in cases where the DNA binding domain binds to its target site constitutively (i.e., without the need for an inducing agent to regulate DNA binding). One example of such a constitutive transactivator is GAL4-VP16 (Sadowski, I. et al.(1988) Nature 335:563-564), composed of the yeast GAL4 DNA binding domain linked to the C-terminal region of herpes simplex virus virion protein 16 (Triezenberg, S. J. et al. (1988) Genes Dev. 2:718-729). In contrast, when the DNA binding domain only binds to its target site in the presence or absence of an inducing agent, an inducible transcriptional activator is created. Examples of such inducible transcriptional activators are TetR-VP16, composed of a bacterial Tet repressor linked to VP16 (which binds to tetO sequences in the absence, but not the presence of tetracycline) (Gossen, M., and Bujard, H. (1992) Proc. Natl. Acad. Sci. U.S.A 89, 5547-5551) and rTetR-VP16, composed of a mutated Tet repressor linked to VP16 (which binds to tetO sequences in the presence but not the absence of tetracycline) (Gossen, M., et al. (1995) Science 268, 1766-1769).
The C-terminal transcriptional activation domain of HSV VP16 has been used frequently as the activator component of transactivator fusion proteins because of its strong capacity to stimulate transcription in eukaryotic cells. It has been shown, however, that overexpression of transcription factors can result in "squelching" (Gill, G., and Ptashne, M. (1988) Nature 334, 721-724), which is seen as a consequence of titrating components of the transcriptional machinery from their respective intracellular pools. For VP16, which is one of the most potent transactivators known, it has been demonstrated that its overexpression, e.g. as a fusion protein with GAL4, is not tolerated by cells (Berger, S. L., et al. (1992) Cell 70, 251-265, Kelleher, R. J., et al. (1990) Cell 61, 1209-1215). Considering that VP16 interacts with a variety of essential components of the transcriptional machinery, including the adaptor/coactivator protein ADA2 in S. cerevisiae (Silverman, N., et al. (1994) Proc. Natl. Acad. Sci. U.S.A 91, 11665-11668) and its human homologue (Candau, R., et al. (1996) Mol. Cell Biol. 16, 593-602), with TFIIB (Lin, Y. S., et al. (1991) Nature 353, 569-571), TFIID (Stringer, K. F., et al. (1990) Nature 345, 783-786), TFIIH (Xiao, H., et al. (1994) Mol. Cell Biol. 14, 7013-7024) and dTAFII40 (Goodrich, J. A., et al. (1993) Cell 75, 519-530), this is not surprising. Gilbert and coworkers (Gilbert, D. M., et al. (1993) Mol. Cell. Biol 13, 462-472) have found a correlation between squelching and growth arrest which indicates that toxicity through squelching is a quantitative problem where the intracellular concentration and the strength of activation domains are crucial parameters.
Thus, while the potent transcriptional activation ability of VP16 makes it an attractive component for use in transactivator fusion proteins, in certain instances it may be desirable to have a fusion protein with a lower transcriptional activation potential than that provided by wild type VP16. Alternatively, in other situations, it may be desirable to have a fusion protein with an even higher transcriptional activation potential than that provided by wild type VP16. Accordingly, additional transactivator fusion proteins with graded transactivation potentials are needed.